中藥蓮子在肝硬化鼠的療效研究

黃怡超
國立陽明大學
因慢性B或C型肝炎及酗酒性肝炎等所致慢性肝臟損傷、發炎有可能導致肝硬化，特徵為肝正常組織及細胞漸被結疤組織所取代，即肝纖維化。慢性B或C型肝炎併有肝硬化是導致肝癌之重要危險因子，故如何阻延或逆轉肝纖維化為處理慢性B或C型肝炎及預防肝癌的重要課題。肝纖維化是一個動態、進行性的過程，在某些階段為可逆的。而對肝纖維化目前尚無有效藥物治療，醫學界仍致力實驗研究，尋找阻延肝纖維化之藥物。
肝纖維化之特徵為肝臟星狀細胞（hepatic stellate cells)”活化”， 即星狀細胞由靜態儲存維生素A細胞轉化為具有增生，收縮肝小竇，以及分泌細胞外間質等特性而促進肝纖維化；如何逆轉成緩解肝纖維化是國際熱門研究課題，目前並無”抗肝纖維化”之西藥，是以中藥之研發有其意義與契機，特別針對是肝纖維化之分子形成機轉，包括:氧化壓力、肝星狀細胞增生與移行、發炎反應等等。中藥蓮子（Nelumbo nucifera)在”神農本草經”記載味甘、澀、平;歸心、肝、胃經。現代研究顯示蓮子萃取物具有抗氧化壓力，清除活性氧化物，抑制紅斑性狼瘡腎炎（lupus nephritis) 之效應。本研究室初步結果顯示蓮子萃取物在離體肝臟星狀細胞具有抑制纖維化之作用，在開發治療抗肝纖維化之中藥生技研發上，有其意義。本計畫擬以（1）離體細胞（包括肝臟星狀細胞），與（2）活體肝纖維化動物模式，建立各種生物活性評估方法後，針對蓮子萃取物與有效成份，在離體及活體上之抗肝纖維化及保護肝細胞之效應做研究，並進而探究其藥效機轉。肝星狀細胞經SV40病毒轉染之細胞株(HSC-T6)，分別以各種刺激因子（血小板源生長因子PDGF，乙型轉型生長因子TGF-β1或腫瘤壞死因子TNF-a）等引致其活化、增生、分泌膠原蛋白、細胞移行等等，再以清除自由基或抑制星狀細胞活化的藥物來評估其效應。有關對肝星狀細胞之抑制是否透過訊息傳遞途徑（例如轉錄因子NFkB，MAP Kinases-ERK/JNK/p38, AP-1，Smad等）或細胞凋亡途徑（核染色質濃縮、斷裂特徵，caspases 3, 8, 9）之細胞分子生物學標記，將依標準方法測定。活體動物效應研究將以兩種肝硬化動物模式來評估其療效。以膽管結紮及肝毒素dimethylnitrosamine引致肝纖維化後，以不同劑量及療程的蓮子萃取物餵食以做療效評估—包括病理切片肝纖維化嚴重度、肝中膠原蛋白與a-SMA含量測定，免疫螢光染色(NFkB與a-SMA ) ，肝纖維化細胞分子生物學標記（TGF-β1，pro-collagen第Ⅰ、III型，metalloproteinases， iNOS等訊息RNA之表現），肝細胞與星狀細胞之氧化壓力指標（NADPH氧化酶訊息傳遞途徑，MDA，4-hydroxynonenal，8-OHdG，glutathione，粒線體通透電位mitochondrial permeability transition），血清生化指標（SGOT，SGPT，creatinine），肝切片中細胞凋亡程度等等。
關鍵字：肝纖維化，中藥，蓮子，肝星狀細胞

Therapeutic Study of a Chinese Herb Nelumbo nucifera on Cirrhotic Rats

Yi-Tsau Huang
National Yang-Ming university
Many patients with chronic viral or alcoholic hepatitis are in the state of chronic liver injury and inflammation and may lead to cirrhosis, with replacement of normal parenchyma by fibrotic tissue. Chronic viral hepatitis B and C are well-recognized risk factors for hepatoma, therefore lessening or reversing hepatic fibrosis is an important agenda for both prevention of hepatoma and management of hepatitis sequelae. Fibrogenesis is a dynamic, complex, and progressive process and at some stages, it is reversible. No clinically efficacious anti-fibrotic drugs are yet available, and current research is directed to lessening or reversing fibrosis.
The major culprit of liver fibrosis is activation of hepatic stellate cells (HSCs), characterized by transformation from a quiescent phenotype to a proliferative, contractile and secretory phenotype. Nelumbo nucifera（蓮子) is a common edible and medicinal plant used in Asia for the treatment of diarrhea, bleeding, fever, and infection. Extracts from Nelumbo nucifera have been shown to exert antioxidant, free radical scavenging activities with anti-inflammatory effects on lupus nephritic mice. Our preliminary results also indicate Nelumbo nucifera extract exerts in vitro anti-fibrogenic effects in HSCs without cytotoxicity. In this study, we will use: (1) in vitro cell assays (HSCs) and (2) in vivo rat models of fibrosis to investigate the anti-fibrotic effects of Nelumbo nucifera extract and elucidate its mechanisms of action. A cell line (HSC-T6) is challenged with cytokines or growth factors to induce HSC activation, and to evaluate its inhibitory effects on activated HSCs. The possibilities of HSC inhibition via signaling cascades (e.g., NF-kB, MAP kinases-ERK/JNK/p38, AP-1, Smads) or apoptotic pathways (caspases 3, 8, or 9) are examined by standard assays. Two rat models of cirrhosis are induced by (a) bile duct ligation, and (b) repeated administration of 1% dimethylnitrosamine. Nelumbo nucifera extract will each be administered at low (X) and high (3-5 X) doses. Treatment duration will include both short-term and long-term periods. Assessment of therapeutic effects will include: (A) histological scoring of liver fibrosis, (B) quantitation of hepatic collagens and a-smooth muscle actin (a-SMA), (C) immunohistochemical staining of a-SMA and NF-kB, (D) molecular markers of fibrogenesis including mRNA expressions of TGF-b1, pro-collagens I and III, and metalloproteinases, (E) cellular oxidative stress markers such as NADPH oxidase activation pathway, MDA, 4-hydroxynonenal, glutathione and 8-OHdG levels, and mitochondrial permeability transition in liver tissue, (F) serum biochemical markers including SGOT, SGPT, and creatinine, and (G) in situ detection of apoptosis in liver tissue.
關鍵字：hepatic fibrosis; Chinese herb; Nelumbo nucifera;hepatic stellate cells